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3 protocols using rp 8 br camps

1

Ginsenoside Rg3 Regulation of Platelet Function

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We purchased 20(S)-ginsenoside Rg3 from the Ambo Institute (Daejon, Korea) and thrombin and all materials for platelet aggregation from Chrono-Log Corporation (Havertown, PA, USA). We purchased all materials for buffer solution and Rp-8-Br-cAMPS, Rp-8-Br-cGMP, p-chlorophenylthio (CPT)-cAMP, 8-Br-cGMP from Sigma (St. Louis, MO, USA) and Fura 2-acetoxymethyl (AM) from Invitrogen (Eugene, OR, USA). Thapsigargin and cAMP/cGMP enzyme immunoassay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-IP3-receptor type I, anti-phosphor-IP3-receptor type I (Ser1756), anti-extracellular signal-regulated kinase (ERK) (1/2), anti-phosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase, and cell lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK).
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2

Modulating IL-2 Expression in Jurkat Cells

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Jurkat E6 cells expressing an IL-2::Luciferase reporter construct (44 (link)) were either pre-treated for 30 min with the PKA inhibitors Rp-8-Br-cAMPS (200 nM) or H89 (1μM; both Sigma Aldrich) or transduced with an empty control-vector or the human PDE4A cDNA. 2 × 106 cells were activated with PMA (1 × 10−7 M, Sigma Aldrich) and PHA (12.5 μg/mL; Thermo Scientific, Waltham, MA). After 6 h, cells were lysed and luciferase activity was on a GloMax 96 well luminometer (Promega, Madison, WI).
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3

PACAP and VIP Signaling Mechanisms

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PACAP (38 amino acid form) and VIP were purchased from Peptide Institute Inc.
(Osaka, Japan). Max was kindly donated by Dr. M Tajima (Shiseido, Japan). DAB, GF109203X and phorbol 12-myristate 13-acetate (PMA) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Forskolin and AR-C155858 were from Merck Millipore (Darmstadt, Germany). H89 was from Seikagaku Co. (Tokyo, Japan).
L-lactate and Rp-8-Br-cAMPS were from Sigma-Aldrich Co. LLC (St. Louis, MO). this study were determined according to our previous reports 17, 27, 28 .
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